This invention relates to a process for producing a heat-resistant acetate kinase.
Acetate kinase is an enzyme widely used for the synthesis of adenosine triphosphate, a source of biological energy, from acetylphosphoric acid and adenosine diphosphate, or for determination of the acetic acid levels in foods. Acetate kinase is usually obtained as a purified enzyme from Escherichia coli (see Journal of Biological Chemistry, 211, p. 737, 1954, and Methods in Enzymology, 1, p. 591), but the acetate kinase isolated from Escherichia coli is very unstable and not suitable for use on an industrial scale.
Japanese Patent Application (OPI) No. 25088/77 (the term "OPI" as used herein means a published unexamined Japanese patent application) describes a process for producing a highly heat-resistant (i.e., heat-stable) acetate kinase from a thermophilic bacterium Bacillus stearothermophilus. But since the acetate kinase produced is an endoenzyme, cultured cells must be first collected by centrifugation or other suitable means before the desired enzyme can be extracted from the cells by ultrasonic treatment or physical breaking.
Since bacterial cells are smaller in size than yeast or fungal cells, they are difficult to collect from the culture, so they are usually collected by centrifugation rather than by filtration. But the rate of sedimentation of cells in centrifugation is proportional to the square of the cell diameter (see Sekiyu Hakko, Petroleum Fermentation, p. 102, 1970, Saiwai Publishing Company), so the recovery of bacterial cells from the fermentation broth is more difficult and expensive than recovery of yeast and fungal cells, and is very disadvantageous in an industrial operation. According to the estimate by Daniel I. C. Wang, the cost of recovery of bacteria is about 3.8 times the cost of recovery of yeast (see Chemical Engineering, Vol. 15, p. 99, 1968).
Therefore, several methods have been proposed for improving the recovery of bacterial cells. Among the proposed methods is a method including flocculation of cells with a flocculant such as ferric chloride, calcium chloride or polymeric flocculant, and a method of modifying the cell protein to an easily collectable form by heating or treatment with a strong acid or base. These techniques are effective when the cell is not the end product, but when the cell per se or the components in the cell are the end product, they are not effective because the flocculant contaminates the product, or the components in the cell are denatured. Furthermore, bacteria belonging to the genus Bacillus have a relatively hard cell wall which can be broken only slightly. Therefore, the efficiency in extraction of intracellular components is low. For these reasons, the techniques proposed to date for recovery of bacterial cells are not suitable for use in industrial large-scale production.